The present invention relates to a flexible cryopreservation bag assembly and method for the freezing, storing, and transferring of mammalian cells. These mammalian cells are used to inoculate a bioreactor.
Biotechnology products typically involve introducing some genetic modification into mammalian cells and then growing these cells in a bioreactor. These bioreactors are specially designed to allow the growth and maintenance of mammalian cells.
For the production of a drug from a specific line of genetically modified mammalian cells, a uniform cell source is needed every time a bioreactor is inoculated. This is the reason for a Manufacturer""s Working Cell Bank (MWCB). An MWCB consists of many aliquots (portions) of a cell suspension, each containing the same type of cells and approximately the same number of cells. These aliquots are prepared on the same day and frozen at the same time. The aliquots are then kept at very cold temperatures (cryopreserved). For each run, one of these aliquots of cells is thawed to provide the same starting point as any other run with the same cells.
Successful inoculation of a bioreactor with mammalian cells requires a minimum cell density to achieve proper cell growth. If the cell density is below the minimum level, extended time requirements are required to achieve commercial cell growth levels, which adds expense to the process and the increases the opportunity for contaminants to enter the cell environment. If the cell density is too high, the nutrients in the media can be depleted, which results in reduced cell growth, and possibly death of the cell culture.
Under current practice, each aliquot in an MWCB has a volume of only 1-2 mL. This limited volume is necessitated by the need to freeze all cells in a rapid and uniform manner. Using existing technology, if large volumes are frozen, the diameter of the suspension will impact the rate of freezing and thawing of the cells, which can damage the cells and adversely impact bioreactor inoculation. The cell concentration in each aliquot is usually in the range of 1-20 million cells/mL. Production bioreactors typically contain tens of liters, hundreds of liters, or even greater volumes. Because of this dramatic difference in volume, a xe2x80x9cseed train expansionxe2x80x9d is used to increase the number of cells until there are enough to introduce into the production bioreactor. For all of the above reasons, the seed train expansion for a production campaign is a critical process step.
In typical production protocols, an aliquot of cells from the MWCB is thawed and the cells are cultured in small bottles (such as T-flasks). As the cells multiply, they are transferred to increasing numbers of small bottles. This increases the total number of cells without having too high or low a cell density. Once enough cells have accumulated, they are transferred into larger bottles (such as roller bottles or shake flasks). When another threshold is reached, the cells are transferred to an inoculation bioreactor. Finally, when enough cells have accumulated in the inoculation bioreactor, they are transferred to a production bioreactor. The seed train described is general practice for several mammalian cell lines and is widely used in commercial production and academia (Lindl and Bauer, 1989, Morgan and Darling, 1993). An overview of a commercial seed train expansion using T-flasks and spinner flasks is also given by Whitaker et al. 1998.
Typically, screw-top vials are used for the MWCB. The uses of screw-top vials necessitates unscrewing the vial caps prior to filling and screwing them back on after filling is a cumbersome manual operation. What is of greater concern is that while vials are open, the contents are subject to contamination. It is extremely important that no contamination is introduced into the vials.
Seed train expansions typically take about four to six weeks to complete under optimal conditions. Because of the large number of transfer steps, seed trains are labor intensive and are susceptible to contamination. Additionally, the environment in the small bottles and larger bottles are not well instrumented or controlled. Changes in pH and oxygen levels may lead to heterogeneity in the cells that are transferred to the production bioreactor. This in turn may lead to variability from one bioreactor run to another and ultimately can impact the quality of the drug being produced.
Each of the above-described risks to the preparation of a homogenous and uniformly viable cell suspension explains the need for improvements in this technology.
The invention is a novel cell freezing and storage bag assembly and method for using the assembly in the seed train expansion of mammalian cells. The bag is constructed principally of polytetrafluoroethylene fabric. The bag is designed to hold enough cells that a bioreactor can be inoculated directly. The bag is designed to be filled to a fraction of its maximum capacity so that the cell suspension has a very thin cross-section. The bag design includes a transfer set that can be sterilely welded to the source of the mammalian cells. This sterile-weldable transfer set allows the bags to be filled quickly with minimal risk of contamination. In the method, once each bag is filled, it is sealed below the connection with the transfer set and the bag is cut xe2x80x9cabovexe2x80x9d the new seal (on the same side of the seal as the transfer set). When a bioreactor is to be inoculated, the contents of the bag are drained via a sterile-weldable inoculation line. During freezing and storage, the inoculation line is protected from mechanical damage.